Substituted N-ethylglycine derivatives for preparing PNA and PNA/DNA hybrids
The present invention relates to novel substituted N-ethylglycine derivatives for preparing PNA and PNA/DNA hybrids as described in the simultaneously filed application xe2x80x9cPeptide oligonucleotide derivatives, their preparation and their usexe2x80x9d (HOE 94/F 057, DE-P 44 08 534.6)
Peptide or polyamide nucleic acids (PNA) are DNA-analogous compounds, in which the deoxyribose phosphate backbone was replaced by a peptide oligomer. The syntheses hitherto described in the literature (for example Michael Egholm, Peter E. Nielsen, Rolf H. Berg and Ole Buchardt, Science 1991, 254, 1497-1500; Ole Buchardt, Michael Egholm, Peter E. Nielsen and Rolf H. Berg, WO 92/20702) use, as a temporary protective group for the amino group of the monomer, the acid-labile tert-butyloxycarbonyl (Boc) protective group, which is eliminated by medium-strong acids such as, for example, trifluoroacetic acid. The solid-phase synthesis of oligomers is carried out in accordance with the customary peptide synthesis processes, as they have been described, for example, by Merrifield (B. Merrifield, J. Am. Chem. Soc., 1963, 85, 2149). The PNA oligomer is eliminated with the aid of a strong acid, customarily using liquid hydrogen fluoride.
The repeated treatment with trifluoroacetic acid and the subsequent cleavage using hydrogen fluoride is not compatible with the synthesis of mixed PNA/DNA sequences, since the nucleosidic linkage is not stable under these conditions. In particular, the purine nucleotides deoxyguanosine and deoxyadenosine are rapidly cleaved on the N-glycosidic linkage by strong acids. Moreover, it would be particularly desirable for the synthesis of such molecules to use the customary DNA synthesizers and to largely retain the chemistry used in this apparatus. This also applies to the preparation of PNA sequences with the aid of such apparatus.
It is therefore an aim of the invention to provide glycine derivatives which allow a simple construction of PNA and PNA/DNA hybrids as well as the use of automatic synthesizers.
Substances which are suitable for this purpose are the compounds of the formula I 
in which
PG is a urethane-type amino protective group which is labile to weak acids, such as, for example, 1-(1-adamantyl)1-methylethoxycarbonyl (Adpoc), 1-(3,5-di-tert-butylphenyl)1-methylethoxycarbonyl (t-Bumeoc) and 1-methyl-1-(4-biphenyl)ethyloxycarbonyl (Bpoc), 3,5-dimethoxyphenyl-2-propyl-2-oxycarbonyl (Ddz), or a trityl-type amino protective group which is labile to weak acids, such as triphenyl (Trt), (4-methoxyphenyl)diphenylmethyl (Trt), (4-methylphenyl)diphenylmethyl (Mtt), di-(4-methoxyphenyl)phenylmethyl (Dmt) and 9-(9-phenyl)xanthenyl (pixyl),
X is NH, O or S, preferably NH or O,
Y is CH2, NH or O, preferably CH2, and
Bxe2x80x2 are bases customary in nucleotide chemistry, for example natural bases, such as adenine, cytosine, guanine, thymine and uracil, or unnatural bases, such as purine, 2,6-diaminopurine, 7-deazaadenine, 7-deazaguanine, N4N4-ethanocytosine, N6N6-ethano-2,6 diaminopurine, 5-methylcytosine, 5-(C3-C6)-alkynyluracil, 5-(C3-C6)alkynylcytosine, 5-fluorouracil and pseudoisocytosine, the exocyclic amino or hydroxyl groups of all of these being protected by suitable known protective groups, such as the benzoyl, isobutanoyl, acetyl, phenoxyacetyl, 4-(t-butyl)benzoyl, 4-(t-butyl)phenoxyacetyl, 4-(methoxy)benzoyl, 2-(4-nitrophenyl)ethyloxycarbonyl, 2-(2,4-dinitrophenyl)ethyloxycarbonyl, 9-fluorenylmethoxycarbonyl, diphenylcarbamoyl or formamidine group, preferably the benzoyl, isobutanoyl, acetyl, phenoxyacetyl, 4-(tbutyl)benzoyl or 4-(methoxy)benzoyl group, and also, in the case of guanine, by a combination of 2-N-acetyl with 6-O-diphenylcarbamoyl, or are base substitute compounds, such as, for example, imidazole, triazole or nitroimidazole, and their salts, preferably their salts with tertiary organic bases, such as, for example, triethylamine or pyridine.
Compounds of the formula I where Y is CH2 can be obtained, for example, by reacting a compound of the formula II
in which
PG and X are as defined above and,
R1 is hydrogen or an ester protective group, such as, for example, methyl, ethyl, butyl or 2-(methoxyethoxy)ethyl,
with a compound of the formula III 
in which
Bxe2x80x2 is as defined above and
Y is CH2 
at 0-45xc2x0 C., preferably at room temperature, in a suitable solvent, such as, for example, DMF, acetonitrile, dichloromethane or mixtures of these solvents, using a coupling reagent conventionally used in peptide chemistry, such as, for example, carbodiimides, phosphonium reagents, uronium reagents, acid halides or activated esters, to give a compound of the formula IV 
in which PG, X, Bxe2x80x2 and R1 are as defined above and subsequently converting this compound to a compound of the formula I by eliminating the ester protective group R1 under weakly alkaline conditions using alkali metal hydroxide solution, such as, for example, NaOH, LiOH, KOH, or by tertiary amine compounds in water, such as, for example, triethylamine, or else enzymatically with the aid of esterases or lipases at 0-50xc2x0 C., preferably at room temperature, in a suitable solvent, such as dioxane, water, tetrahydrofuran, methanol, water or mixtures of the solvents.
Activation methods conventionally used in peptide synthesis are described, for example, in Houben-Weyl, Methoden der organischen Chemie [Methods in organic Chemistry], Volume 15/2, Georg Thieme Verlag Stuttgart 1974, or further reagents are described in the particular references, for example BOP (B. Castro, J. R. Dormoy, G. Evin and C. Selve, Tetrahedron Lett. 1975, 1219-1222), PyBOP (J. Coste, D. Le-Nguyen and B. Castro, Tetrahedron Lett. 1990, 205-208), BroP (J. Coste, M.-N. Dufour, A. Pantaloni and B. Castro, Tetrahedron Lett. 1990, 669-672), PyBroP (J. Coste, E. Frerot, P. Jouin and B. Castro, Tetrahedron Lett. 1991, 1967-1970) and uronium reagents, such as, for example, HBTU (V. Dourtoglou, B. Gross, V. Lambropoulou, C. Zioudrou, Synthesis 1984, 572-574), TBTU, TPTU, TSTU, TNTU, (R. Knorr, A. Trzeciak, W. Bannwarth and D. Gillessen, Tetrahedron Letters 1989, 1927-1930), TOTU (EP-A-0 460 446), HATU (L. A. Carpino, J. Am. Chem. Soc. 1993, 115, 4397-4398), HAPyU, TaPipU (A. Ehrlich, S. Rothemund, M. Brudel, M. Beyermann, L. A. Carpino and M. Bienert, Tetrahedron Lett. 1993, 4781-4784), BOI (K. Akaji, N. Kuriyama, T. Kimura, Y. Fujiwara and Y. Kiso, Tetrahedron Lett. 1992, 3177-3180) or acid chlorides or acid fluorides (L. A. Carpino, H. G. Chao, M. Beyermann and M. Bienert, J. Org. Chem., 56(1991), 2635; J.-N. Bertho, A. Loffet, C. Pinel, F. Reuther and G. Sennyey in E. Giralt and D. Andreu (Eds.) Peptides 1990, Escom Science Publishers B. V. 1991, pp. 53-54; J. Green and K. Bradley, Tetrahedron 1993, 4141-4146), 2,4,6-mesitylenesulfonyl-3-nitro-1,2,4-triazolide (MSNT) (B. Blankemeyer-Menge, M. Nimitz and R. Frank, Tetrahedron Lett. 1990, 1701-1704), 2,5-diphenyl-2,3-dihydro-3-oxo-4-hydroxythiophene dioxide (TDO) (R. Kirstgen, R. C. Sheppard, W. Steglich, J. Chem. Soc. Chem. Commun. 1987, 1870-1871) or activated esters (D. Hudson) Peptide Res. 1990, 51-55).
Preferred is the use of carbodiimides, for example dicyclohexylcarbodiimide or diisopropylcarbodiimide. Other reagents which are preferably used are phosphonium reagents, such as, for example, PyBOP or PbBroP, uronium reagents, such as, for example HBTU, TBTU, TPTU, TSTU, TNTU, TOTU or HATU, BOI or acid chlorides or acid fluorides.
To synthesize the compounds of the formula II, aminoethylglycine, hydroxyethylglycine, mercaptoethylglycine or their corresponding esters are provided with the corresponding protective group which is labile to weak acids. The protective group which is labile to weak acids is introduced with the aid of processes per se known from the literature, some of which have been modified. Examples of suitable reagents are t-Bumeoc fluoride, Adpoc azide, Bpoc azide, Ddz (phenyl)carbonate, Trt Cl, Mtt Cl, Mmt Cl, Mmt Cl, Dmt Cl, Pixyl Cl. In this reaction, the solubility of the aminoethylglycine can be improved while simultaneously protecting the acid function by reacting it with customary silylation reagents, such as, for example, bis-trimethylsilylacetamide. After the reaction with the protective group reagents, this temporary protective group is eliminated by adding water or alcohols to the reaction mixture. The aminoethylglycine or the corresponding aminoethylglycine ester used as the starting material are prepared by a method known from the literature (E. P. Heimer, H. E. Gallo-Torres, A. M. Felix, M. Ahmad, T. J. Lambros, F. Scheidl and J. Meienhofer, Int. J. Peptide Protein Res. 23, 1984, 203-211) 2-aminoethylglycine (H-Aeg-OH).
A further process for the preparation of aminoethylglycine consists in subjecting glyoxylic acid to reductive amination with ethylene diamine and is described in the application titled xe2x80x9cProcess for the preparation of aminoethylglycinexe2x80x9d (HOE 94/F 061, DE-P 44 08 530.3) filed simultaneously.
2-Hydroxyethylglycine or 2-mercaptoethylglycine is synthesized, for example, by subjecting glyoxylic acid or glyoxylic esters to reductive amination with aminoethanol or cysteamine using hydrogen on palladium-on-charcoal or, in the case of 2-mercaptoethylglycine, preferably using sodium cyanoborohydride or sodium triacetoxyborohydride as the reducing agent.
The compound of the formula I, which is derived from 2-mercaptoethylglycine, can also be obtained by first reacting 2-mercaptoethylamine or its hydrochloride with the corresponding triphenyl derivative, such as, for example, its halides or alcohols, in acetic acid or mixtures of acetic acid/water. Under the acidic conditions, the reaction is preferably effected on the sulfur of 2-mercaptoethylamine via the corresponding trityl cation. The use of (4-methoxyphenyl)diphenylmethyl chloride is preferred for introducing the Mmt group, and the use of di-(4-methoxyphenyl)phenylmethyl chloride for introducing the Dmt group. The amino group is subsequently alkylated by reaction with haloacetic esters, preferably bromoacetic esters, using an organic auxiliary base, such as, for example, diisopropyl ethyl amine, in a suitable solvent, such as, for example, DMF. The resulting compound of the formula II in which PG is a trityl-type protective group, X is S and R1 is an ester protective group can then be reacted in the manner described above to give the corresponding compounds of the formula I.
The acetic acid derivatives of the nucleobases, of the formula III, can be retained by alkylating the corresponding nucleobases or the nucleobases which are protected in the exocyclic hydroxyl or amino function using chloroacetic acid, bromoacetic acid, iodoacetic acid, or their esters. For preference, temporary protective groups are additionally introduced on the nucleobase for the purposes of selective alkylation. Protective groups which are suitable for the nucleobases are all protective groups which are compatible with the protective group PG which is labile to weak acids. Protective groups which are preferably used for the exocyclic amino function are, for example, the benzoyl, isobutanoyl, acetyl, phenoxyacetyl, 4-(t-butyl)benzoyl, 4-(t-butyl)phenoxyacetyl, 4-(methoxy)benzoyl, 2-(4-nitrophenyl)ethyloxycarbonyl, 2(2,4-dinitrophenyl)ethyloxycarbonyl, 9-fluorenylmethoxycarbonyl, diphenylcarbamoyl, or formamidine group.
Particularly preferred are the benzoyl, isobutanoyl, 4-(t-butyl)benzoyl, 2-(4-nitrophenyl)ethyloxycarbonyl, 2(2,4-dinitrophenyl)ethyloxycarbonyl, 9-fluorenylmethoxycarbonyl, 4-(methoxy)benzoyl or para-(t-butyl)phenoxyacetyl or para-nitrophenyl-2-ethyloxycarbonyl group and, in the case of guanine, a combination of the 2-N-acetyl with the 6-O-diphenylcarbamoyl group.
An alternative process for the preparation of the compounds of the formula I in which Y is CH2 consists in reacting
a compound of the formula II 
in which
PG and X are as defined above and
R1 is hydrogen or a temporary silyl protective group, such as, for example, trimethylsilyl,
with a compound of the formula V
Bxe2x80x2xe2x80x94CH2xe2x80x94COxe2x80x94R2xe2x80x83xe2x80x83(V),
in which
Bxe2x80x2 is as defined above and
R2 is halogen, such as, for example, fluorine, chlorine or bromine, or the radical of an active ester, such as, for example, OBt, OObt, OPfp, ONSu,
at 0-40xc2x0 C., preferably 20-30xc2x0 C., in a suitable solvent, such as, for example, DMF, NMP, acetonitrile, dichloromethane or mixtures of these solvents, it optionally being possible to protect the acid function in the compound of the formula II temporarily by reacting it with customary silylation reagents, such as, for example, bis-trimethylsilylacetamide, and to eliminate these temporarily protective groupsxe2x80x94after the reaction with the compound of the formula Vxe2x80x94by adding water or alcohols to the reaction mixture.
A further alternative process for the preparation of the compounds of the formula I where Y is CH2 consists in reacting
a compound of the formula II 
in which
PG and X are as defined above and
R1 is an ester protective group, such as, for example, methyl, ethyl, butyl, 2-(methoxyethoxy)ethyl and the like,
with a haloacetic acid derivative, such as, for example, chloroacetyl chloride, bromoacetyl bromide, bromoacetyl chloride or iodoacetyl chloride, in a suitable solvent, such as, for example, tetrahydrofuran, dichloromethane or DMF, using an auxiliary base, such as, for example, triethylamine, N-ethylmorpholine or diisopropylethylamine, to give the compound of the formula VI 
in which
Hal is Cl, Br or I, preferably Br or Cl, very particularly Cl, and
PG, X and R are as defined above,
reacting this intermediate of the formula VI with the optionally protected nucleobase Bxe2x80x2 and an auxiliary base, for example potassium carbonate, in a suitable solvent, for example DMF or NMP, to give the compound of the formula IV 
and subsequently converting this compound to a compound of the formula I by eliminating the ester protective group R1 using alkali metal hydroxide solution, such as, for example, NaOH, LiOH or KOH, or else enzymatically with the aid of esterases or lipases at 0-50xc2x0 C., preferably at room temperature, in a suitable solvent, such as dioxane, water, tetrahydrofuran, methanol, water or mixtures of these solvents.
Compounds of the formula I in which Y is o or NH are obtained by reacting
a compound of the formula II 
in which
PG and X are as defined above and
R1 is an ester protective group, such as, for example, methyl, ethyl, butyl or 2-(methoxyethoxy)ethyl, or a temporary ester protective group, such as, for example, trimethylsilyl,
with a compound of the formula VII
Bxe2x80x2xe2x80x94Yxe2x80x94COxe2x80x94R3xe2x80x83xe2x80x83(VII)
in which
Bxe2x80x2 is as defined above,
Y is O or NH and
R3 is Cl, ONp or ONSu
at 0-40xc2x0 C., preferably 20-30xc2x0 C., in a suitable solvent, such as DMF, acetonitrile or dichloromethane, or mixtures of these solvents, and subsequently eliminating the ester protective group R1 using alkali metal hydroxide solution, such as, for example, NaOH, LiOH or KOH, or else enzymatically with the aid of esterases or lipases at 0-50xc2x0 C., preferably at room temperature, in a suitable solvent, such as dioxane, water, tetrahydrofuran, methanol, water or mixtures of these solvents.
The N-amino- or N-hydroxy-nucleobases required as starting material for this latter reaction are obtained by known processes, such as, for example, as described by K. Kohda, I. Kobayashi, K. Itano, S. Asano, Y. Kawazoe (Tetrahedron 49, 3947-3958 (1993)) and then reacted with phosgene or chloroformic esters to give the carbamates or carbonates of the formula VII.
The abbreviations used for amino acids correspond to the three-letter code conventionally used in peptide chemistry, as it is described in Europ. J. Biochem. 138, 9 (1984). Other abbreviations used are listed hereinbelow.